Project Summary A microfluidic-based screening method will be developed to differentiate different disease states of cancer. The screening method is based on high-resolution microchip electrophoresis coupled with laser-induced fluorescence detection. N-Glycan profiles are generated for all samples and compared by statistical analysis. The method relies on the analysis of the entire N-glycan profile, not a single biomarker, to provide sufficient differentiation among control individuals, patients suffering from various stages of cancer, and patients with pre-malignant diseases. Microchip electrophoresis with fluorescence detection provides excellent resolution of glycan structures and their isomers and has tremendous detection sensitivity. Preliminary results suggest that separation channels e20 cm in length and separation field strengths e750 V/cm are able to rapidly and efficiently separate N-glycans derived from clinically relevant serum samples, e.g., from patients with breast cancer, ovarian cancer, and esophageal adenocarcinoma. Efficient separation and sensitive detection on capillary- or chip-based instruments permit both low and high abundance N-glycans to contribute to the statistical analysis for disease-state differentiation. In this application, microfluidic devices will be used as a convenient and reliable platform to screen N-glycans associated with cancer. As microfluidic-based methods are optimized, those N-glycans that contribute most to disease-state differentiation will be identified. Determination of these molecular structures will enable optimization and validation of the screening method through improvements to electrophoretic analysis and detection sensitivity. The specific aims of this application are to: (1) demonstrate that microchip electrophoresis of N- glycans is a rapid, reliable screening method for various cancers, (2) develop a suite of N- glycan standards to be used as sizing ladders for standard addition in microchip electrophoresis, (3) determine N-glycan structures by mass-spectrometric analysis, and (4) integrate monolithic phases into the microfluidic devices for dialysis, solid-phase extraction, electrochromatography, and enzymatic sequencing.